Interpretive Summary: The U.S. Food and Drug Administration (FDA) utilizes the presence of filth and extraneous materials as one of the criteria for implementing regulatory actions and assessing adulteration of food products of public health importance. Twenty-two prevalent pest species (also known as the ‘‘Dirty 22’’ species) have been considered by the FDA as possible vehicles for the spread of foodborne diseases, and the presence of these species is considered an indicator of unsanitary conditions in food processing and storage facilities. Scientists from the FDA Southeast Regional Laboratory and the USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology described the development of three novel nested PCR primer sets and multilocus genetic characterization for the Pharaoh ant and the thief ant, two species among the “Dirty 22”. These species-specific nested PCR primer sets can be used when the specimens cannot be identified using conventional microscopic methods and will be useful in the control of foodborne pathogens.

Technical Abstract:
The U.S. Food and Drug Administration utilizes the presence of filth and extraneous materials as one of the criteria for implementing regulatory actions and assessing adulteration of food products of public health importance. Twenty-two prevalent pest species (also known as the ‘‘Dirty 22’’ species) have been considered by this agency as possible vehicles for the spread of foodborne diseases, and the presence of these species is considered an indicator of unsanitary conditions in food processing and storage facilities. In a previous study, we further categorized the Dirty 22 species into four groups: group I includes four cockroach species, group II includes two ant species, group III includes 12 fly species, and group IV includes four rodent species. Here, we describe the development of three nested PCR primer sets and multilocus genetic characterization by amplifying the small subunit rRNA, elongation factor 1-alpha, and wingless (WNT-1) genes of group II Dirty 22 ant species Monomorium pharaonis and Solenopsis molesta. These novel group II Dirty 22 species-specific nested PCR primer sets can be used when the specimens cannot be identified using conventional microscopic methods. These newly developed assays will provide correct identification of group II Dirty 22 ant species, and the information can be used in the control of foodborne pathogens.